Design, Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

Article abstract of DOI:10.1002/cmdc.202000984

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for thei

Full text of DOI:10.1002/cmdc.202000984

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Design, Synthesis, and Biological Evaluation of Novel
Fluorescent Probes Targeting the 18-kDa Translocator
Protein
Hendris Wongso,*[a, b] Tomoteru Yamasaki,[c] Katsushi Kumata,[c] Maiko Ono,[d]
Makoto Higuchi,[d] Ming-Rong Zhang,[c] Michael J. Fulham,[e] Andrew Katsifis,*[e] and
Paul A. Keller*[a]
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A series of fluorescent probes from the 6-chloro-2-phenyl-
imidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-
nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized
and biologically evaluated for their fluorescence properties and
for their binding affinity to the 18-kDa translocator protein
(TSPO). Spectroscopic studies including UV/Vis absorption and
fluorescence measurements showed that the synthesized
fluorescent probes exhibit favorable spectroscopic properties,
especially in nonpolar environments. In vitro fluorescence
staining in brain sections from lipopolysaccharide (LPS)-injected
mice revealed partial colocalization of the probes with the
TSPO. The TSPO binding affinity of the probes was measured on
crude mitochondrial fractions separated from rat brain homoge-
nates in a [11C]PK11195 radioligand binding assay. All the new
fluorescent probes demonstrated moderate to high binding
affinity to the TSPO, with affinity (Ki) values ranging from
0.58 nM to 3.28 μM. Taking these data together, we propose
that the new fluorescent probes could be used to visualize the
TSPO.
Introduction
to inflammatory stimuli or brain injury.[3,4] It is therefore
considered an important and sensitive marker of microglial-
astrocyte activation and inflammation. In addition to its over-
expression in neurodegenerative diseases, an increase in the
expression of the TSPO has also been seen in atherosclerosis,
stroke, arthritis, cardiovascular disease[5] and several cancers
including brain,[6] prostate,[7] colon,[8] and breast.[9] Furthermore,
it is reported that TSPO expression correlated positively with
disease progression whilst the prognosis of some cancers
correlate negatively with the expression of TSPO.[10] Therefore
numerous studies involving TSPO radioligands have been
investigated as a means of monitoring neuroinflammation,
neurodegeneration, cancer and cardiovascular disease in pre-
clinical and clinical studies using the imaging modalities
positron emission tomography (PET) and single photon emis-
sion computed tomography (SPECT).[11,12]
To date, radioligands targeting TSPO, exemplified by the
isoquinoline carboxamide ([11C]PK11195),[13] imidazo[1,2-a]
pyridine ([18F]PBR111),[14] 2-aryl-8-oxodihydropurine acetamide
([18F]FEDAC),[15] phenoxyarylacetamide ([18F]DAA1106),[16] arylox-
yanilide ([11C]PBR28),[17] and the tricyclic indole ([18F]GE180)[18]
(Figure 1) have been prepared and evaluated preclinically and
clinically.
The 18-kDa translocator protein (TSPO) is part of a five trans-
membrane hetero-oligomeric complex widely expressed in
peripheral organs such as the heart, kidney, lungs, and steroid-
producing tissues and in lower concentrations in the central
nervous system (CNS).[1,2] In the CNS, TSPO is involved in
neurosteroid synthesis, regulation of mitochondrial function,
and modulation of neuroinflammation in microglial cells and
astrocytes. Importantly, TSPO expression is markedly upregu-
lated in activated microglia and reactive astrocytes in response
[a] Dr. H. Wongso, Prof. P. A. Keller
School of Chemistry and Molecular Bioscience, and Molecular Horizons
University of Wollongong
Wollongong, NSW 2522 (Australia)
E-mail: keller@uow.edu.au
hw765@uowmail.edu.au
[b] Dr. H. Wongso
Center for Applied Nuclear Science and Technology
National Nuclear Energy Agency
Bandung 40132 (Indonesia)
[c] Dr. T. Yamasaki, Dr. K. Kumata, Prof. M.-R. Zhang
Department of Advanced Nuclear Medicine Sciences
National Institute of Radiological Sciences
Chiba 263-8555 (Japan)
In parallel, fluorescence/optical imaging, has been widely
used in in vitro histologic examination of cells and tissue, and in
in vivo imaging studies using small animals. However, due to
photon scattering and light attenuation by biological tissue,
fluorescence imaging is limited by depth penetration and is
unable to provide in vivo quantitative or tomographic informa-
tion in large animals or human subjects.[19] More recently,
fluorescence imaging has gained considerable interest in
improving surgical interventions. As a result, clinical studies that
[d] Prof. M. Ono, Prof. M. Higuchi
Department of Functional Brain Imaging Research
National Institutes for Quantum and Radiological Science and Technology
Chiba 263-8555 (Japan)
[e] Prof. M. J. Fulham, Prof. A. Katsifis
Department of PET and Nuclear Medicine
Royal Prince Alfred Hospital
Camperdown, NSW 2050 (Australia)
E-mail: andrewk@nucmed.rpa.cs.nsw.gov.au
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000984
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Figure 1. Structures of several radiolabeled TSPO ligands.
combined the clinical utility of PET-CT and fluorescence
imaging for improving tumor excision, reduced local recurrence
and for improving patient prognosis have been
investigated.[20,21] Hence, the conjugation of various fluoro-
phores to TSPO ligands to produce TSPO fluorescent probes
offers considerable opportunities in biological and clinical
studies to complement the use of radioligands.
The development of high-affinity and selective fluorescent
or dual-modality PET fluorescent imaging agents based on
small drug-like molecules has been synthetically challenging
due to the relatively large size of the fluorescent moiety, and
the steric impact these structures have on the binding to the
receptor. Furthermore, the type of fluorophore, and the mode
of attachment not only impacts affinity and selectivity, but also
biodistribution, uptake and clearance of the imaging agent
in vivo.[22] A common approach to developing fluorescent
probes has been the attachment of the fluorophore to the
parent ligand through the use of linkers to minimize interfer-
ence between the binding domain of the targeting ligand and
the biological target.[23] It is also well recognized that the length
linkers on the acetamide side chain of the imidazopyridine
skeleton which showed moderate affinity for the TSPO.[25]
The aims of this project were to develop high affinity and
selective TSPO fluorescent ligands based on the imidazopyr-
idine chemical structure suitable for in vitro, ex vivo and in vivo
applications. To this end, this work investigated the attachment
of the fluorophore both on the acetamide side chain (fluo-
rophore attachment I) and on the 2-(4’-phenyl) (fluorophore
attachment II) of the 2-phenylimidazo[1,2-a]pyridine-3-yl
acetamide and their preliminary biological evaluation. The
probe incorporated was the simple and relatively small 7-nitro-
2-oxa-1,3-diazole using different sized linkers to study the effect
of these substituents on TSPO binding, the outcomes of which
could be used later to develop other fluorescent probes with
emissions suitable for in vitro, ex vivo or in vivo applications.
Herein, we report and discuss the synthesis and biological
evaluation of these novel imidazopyridine fluorescent probes
for potential use in visualizing the TSPO.
and types of linkers have an enormous influence on the ligand’s Results and Discussion
binding affinity, selectivity, and the pharmacokinetic and
pharmacodynamic properties in vivo.[24,25] The structures of
several fluorescent TSPO probes developed for the visualization
of activated microglia by fluorescence microscopy are shown in
Figure 2.
We have previously identified highly potent and selective TSPO
ligands based on the imidazo[1,2-a]pyridines, pyrozolopyrimi-
dine structures, which were developed as PET ligands radio-
labeled with 18F (Figure 4).[34] This current study investigated
TSPO-fluorescent probes generated from the conjugation of
TSPO ligand 2 with the commercially available fluorophore,
NBD, selected as it has been previously investigated as a
fluorophore for spectroscopic and microscopic applications in
biophysical, biochemical, and cell biological studies.[35,36] NBD-
containing ligands typically exhibit a low quantum yield in
aqueous solution and protic environments, but are highly
Recently, several TSPO ligands have being exploited as
fluorescent/near infrared probes for imaging the TSPO in vitro
and in vivo.[27,28,31,32] Literature data and our own results suggest
that 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides of
general structure 1 (Figure 3) exhibit high affinity for the TSPO,
when R1 and R2 are small alkyl groups whilst the para position
‘X’ on the 2-phenyl ring is able to tolerate relatively large
groups.[33] Thus, it was hypothesized that X, R1 and R2
substituents can be used to attach fluorophores using suitable
linkers. Therefore, the derivatisation of the imidazopyridine is
consistent with a previously reported study involving the
attachment of the fluorescent NBD moiety with various sized
fluorescent in
a nonpolar medium or when bound to
membranes or hydrophobic residues in protein pockets. A
further advantage is its relatively small size compared to other
probes, which may have less impact on the affinity and
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Figure 2. Structure of several TSPO fluorescent probes.
acid (AcOH) to give tertiary amine 7 in 69%, followed by
quaternization with methyl iodide (CH3I) at room temperature
to produce the ammonium iodide 8 in 84%. The desired
acetamide 9 was obtained in 71% yield upon treatment with
KCN, followed by hydrolysis under strongly alkaline conditions
(KOH in ethanol) and gave the corresponding imidazopyridine-
acetic acid 10 in 94% yield (Scheme 1).
The imidazopyridineacetic acid 10 then entered divergent
synthesis pathways to generate probes with the attachment of
the fluorophore moiety at the 3-acetamide side chain (path A,
fluorophore attachment I) or on the phenyl ring of the
imidazopyridine structure (path B, fluorophore attachment II).
The synthesis of fluorescent probes 15a–d (path A; Scheme 2)
involved the peptide coupling of the acid 10 with the
appropriate mono-Boc-protected polymethylendiamine 11a–d
in the presence of HOBt, EDCI, and DIPEA generating amides
Figure 3. General structure of imidazo[1,2-a]pyridine TSPO ligands.
Figure 4. Two examples of PET TSPO ligands radiolabeled with fluorine-18.
selectivity of the parent ligand during ligand development
stages.[24]
Chemistry
The synthesis of imidazopyridineacetic acid 10 started with a
standard condensation of commercially available 5-chloropyr-
idin-2-amine 4 and 2-bromo-4’-methoxyacetophenone 5 in
ethanol at reflux to produce imidazopyridine 6 in 73%, which
subsequently underwent a Mannich condensation with aqueous
formaldehyde (H2CO) and dimethylamine (NH(CH3)2) in acetic
Scheme 1. a) NaHCO3, EtOH, reflux, 10.5 h, 73%; b) (CH3)2NH (aq), CH2O,
°
CH3COOH, 55 C, 18 h, 69%; c) CH3I, toluene, RT, 48 h, darkness, 84%; d) KCN,
EtOH, H2O, reflux, 48 h, 71%; e) KOH, EtOH, H2O, reflux, 18 h, 94%.
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°
Scheme 2. a) HOBt, EDCl, DIPEA, DMF, RT, 24 h, 74–96%; b) CF3CO2H, CH2Cl2, RT, 4 h, 85–89%; c) DIPEA, DMF, 0 C – RT, 18 h, darkness, 42–57%; d) NH4OH,
°
MeOH, 0 C – RT, 18 h, darkness, 51%; e) HOBt, EDCl, DIPEA, DMF, RT, 48 h, 62%. 15a–d, 17: fluorescent probes in MeOH (~1.0%, w/v) upon UV irradiation
(λ=365 nm).
12a–d in 74–96% yields. Boc deprotection with CF3CO2H (TFA)
produced free amines 13a–d in 85–89% yields. Condensation
of amines 13a–d with NBDÀ Cl 14 in the presence of DIPEA to
give the desired probes 15a–d in moderate yields (42–57%).
NBD-amine 16 was obtained in 51% yield from the treatment
of NBDÀ Cl 14 with NH4OH in methanol. Synthesis of probe 17
was achieved from direct conjugation of NBD-amine 16 with
imidazopyridineacetic acid 10 in 62% yield, using a similar
procedure for 12a–d.
The synthesis of probes 23a–b (Path B) was accomplished
in a five-step procedure (Scheme 3), starting with demeth-
ylation and hydrolysis of acetamide 9 using HBr in acetic acid to
afford the corresponding carboxylic acid 18 in 63% yield. The
amide 19 was realized in 74% yield by peptide coupling of the
acid 18 with diethylamine in the presence of HOBt, EDCl, DIPEA
in anhydrous DMF. Alkylation of the phenol group with the
appropriate Boc-protected bromoamine 20a–b, produced O-
alkylated derivatives 21a–b in 71–72% yields. Further treatment
°
Scheme 3. a) HBr, CH3COOH, 110 C, 48 h, 63%; b) DEA, HOBt, EDCl, DIPEA, DMF, RT, 24 h, 74%; c) K2CO3, DMF, RT, 48 h, 71–72%; d) CF3CO2H, CH2Cl2, RT, 4 h,
88–92%; e) DIPEA, DMF, 0 C – RT, 18 h, darkness, 42–44%; f) DIPEA, DMF, 0 C – RT, 18 h, darkness, 43%. 23a–b, 24: fluorescent probes 23a–b, 24 in MeOH
(~1.0% (w/v)) upon UV irradiation (λ=365 nm).
°
°
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with CF3CO2H in CH2Cl2 afforded the free amines 22a–b in 88–
92% yields. The synthesis of fluorescent compounds 23a–b was
completed by conjugation of amines 22a–b with NBDÀ Cl 14 in
the presence of DIPEA in 42–44% yields. Synthesis of probe 24
was achieved in 43% yield via direct conjugation of phenol 19
with NBDÀ Cl 14 in the presence of DIPEA in anhydrous DMF.
(Scheme 3).
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Spectroscopic properties of fluorescent probes 23a, 23b
and 24
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The ultraviolet absorption (Figure 5) and the fluorescence
properties (Figure 6) of the selected fluorescent compounds
23a, 23b and 24 were investigated in different environments.
These probes were selected for spectroscopic studies as they
showed excellent binding affinity for the TSPO (Table 3). As
the first step, compounds were dissolved in dimethyl sulfoxide
Figure 6. Effect of the polarity of the medium on the fluorescence of
compounds 23a and 23b at 10 μM in solutions varying from 0 to 75% v/v
dioxane in PBS.
(DMSO), diluted to a final concentration of 10 μM in different
assay solutions from 0% dioxane to 75% v/v dioxane/water
phosphate-buffered saline (PBS). The percentage of DMSO
used in the assay did not exceed 1% of the final volume
solution. In aqueous solution (0% v/v dioxane/PBS), the
spectrum of probe 23a is characterized by two absorptions
maxima at 340 and 490 nm, showing extinction coefficients of
ɛ=11000 and 10400 MÀ 1 cmÀ 1, respectively. When probe 23a
was dissolved in 25% v/v dioxane/PBS, the spectrum is
characterized by two absorption maxima at 330 and 470 nm,
and the increase of the extinction coefficients (ɛ=15800 and
18700 MÀ 1 cmÀ 1, respectively). A further increase in hydro-
phobicity of the medium (50% v/v dioxane/PBS) caused an
increase of the extinction coefficients to 24700 MÀ 1 cmÀ 1 and
24500 MÀ 1 cmÀ 1, respectively. In the PBS solution containing
75% dioxane (v/v), the extinction coefficients remained almost
constant.
Fluorescence staining
To confirm the probe binding to TSPO expressed with
inflammation, in vitro fluorescence staining using brain sec-
tions from lipopolysaccharide (LPS)-injected mice[38] was
carried out. In this study, 4 μg of LPS was injected into the
striatum of the right hemisphere, and the induction of TSPO
Figure 5. Ultraviolet absorption spectra of compounds 23a, 23b, and 24 at
10 μM in solutions varying from 0 to 75% v/v dioxane in PBS.
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expression with LPS injection was confirmed by immunostain-
ing with anti-TSPO antibody (Table 1). Double staining of brain
sections from LPS-injected mice with probes as well as anti-
TSPO antibody illustrated that the green signals derived from
the tested probes and red signals derived from anti-TSPO
antibody were partially colocalized in several probes, including
15a–d and 23a–b (Table 2). These data suggested that the
binding activity of probes to TSPO in the tissue slices of mouse
brain evoked an inflammation under the environment where
high concentrations of probe (17.3–19.2 μM) can react with
TSPO.
The absorption spectra of probe 23b show a similar profile
to that of probe 23a, suggesting that higher hydrophobicity
of the medium can promote the increase of the extinction
coefficient. From this probe, the highest extinction coefficient
(ɛ=19200 MÀ 1 cmÀ 1) was observed in 75% v/v dioxane/PBS
solution at 470 nm. The absorptions maxima of probe 24 was
generally lower compared to both probes 23a and 23b, with
the spectra characterized by two absorptions maxima at 330
and 470 nm, and the highest extinction coefficient (ɛ=
19200 MÀ 1 cmÀ 1) was observed at 470 nm in the PBS solution
containing 75% dioxane (v/v).
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Table 1. Fluorescent images of brain sections from LPS-injected mice stained with anti-TSPO antibody.
Table 2. Binding of probes to TSPO in the brain section from LPS-injected mouse.
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Probes 23a and 23b displayed emission maxima at
with increasing carbon linker length. The moderate binding
affinity of these probes bearing the NBD substituents on the
acetamide side chain of the imidazopyridine is consistent with
those reported by previous investigators.[25] On the other
hand, the attachment of the NBDs on the para-position of the
phenyl ring of the ligand resulted in an excellent binding
affinity as exemplified by probes 23a (Ki =1.35 nM), 23b (Ki =
0.58 nM) and 24 (Ki =1.44 nM), confirming our previous studies
that this position of the 2-phenylimidazopyridine group was
more tolerant of large functional groups in maintaining affinity
for the TSPO receptor. The fluorescent compound bearing a C2
linker (23b) exhibited the highest binding affinity (Ki =
0.58 nM), which was twice as high as compounds 23a and 24,
respectively. To the best of our knowledge, compound 23b is
the highest affinity fluorescent probe among the NBD-based
TSPO fluorescent probes that have been reported. More
interestingly, the affinity of this compound is 6–10 times
higher than its parent ligand analogues (2a–b), indicating that
the introduction of the fluorophore may not always decrease
the affinity of the parent ligand. Although the binding affinity
of these probes to the central benzodiazepine receptor (CBR)
were not determined, the in vitro studies with these probes in
LPS-lesioned mice shows colocalization with anti-TSPO anti-
body in lesions, suggesting selectivity for the TSPO over the
CBR. Further ligand optimization and their specificity and
selectivity for the TSPO over CBR and other targets will be
conducted.
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535 nm, which correspond to the NBD moiety emission (Fig-
ure 6). Interestingly, probe 23b with the longer carbon linker
(n=2) showed a significant increase of fluorescence intensity
compared to probe 23a (n=1). Additionally, decreasing the
polarity of the medium (from 0 to 75% v/v dioxane in PBS)
promoted an approximately tenfold and 32-fold increase in
the fluorescence intensity of probes 23a and 23b, respec-
tively. This suggests that NBD-containing compounds exhibits
a high degree of environmental sensitivity.[36] In contrast,
probe 24 without any carbon methylene linker between the
parent ligand and NBD demonstrated a very low fluorescence
intensity at 500 to 600 nm, probably due to intramolecular
fluorescence quenching.[37] However, an increase in
fluorescence intensity was observed at 410 nm (see Figure S73
in the Supporting Information).
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TSPO binding affinity
The affinity of the newly synthesized compounds for the TSPO
was determined in a competitive binding assay using crude
mitochondrial fraction, centrifuged from rat brain homoge-
nates with [11C]PK11195 as the competitive radioligand. With
PK11195 as the reference standard, the fluorescent probes
showed moderate to high affinity binding to the TSPO, with Ki
values ranging from 0.58 nM for compound 23b and 3.28 μM
for 15d (Table 3)
Overall, fluorescent compounds with the attachment of
the NBDs at the 3-acetamide side chain (Path A, fluorophore Conclusion
attachment I) showed lower affinity for the TSPO (Ki =242.43–
3276.30 nM) compared to probes with the attachment of the
NBDs on the 4’-position of the 2-phenyl ring of the
imidazopyridine structure (Path B, fluorophore attachment II;
Ki =0.58–1.44 nM). Probes 15a–d with carbon linker varying
from 2 to 6, generally showed low to moderate affinity for the
TSPO. In this subset, significant improvement in affinity was
observed by reducing the length of the linkers. Additionally,
compound 17, which represents the fluorescent compound
without a carbon linker demonstrated higher affinity than
probes 15a–d which showed progressive reduction in affinity
A series of novel fluorescent probes based on the 6-chloro-2-
phenylimidazo[1,2-a]pyridine-3-yl acetamide structure has
been developed, bearing the 7-nitro-2-oxa-1,3-diazol-4-yl
(NBD) moiety on the 2-(4’-phenyl) position as well as on the 3-
acetamide side chain. All of the new fluorescent probes
demonstrated moderate to high binding affinity (Ki) values for
the TSPO, with nanomolar binding of ligands bearing the NBD
in the 2-(4’-phenyl) position compared to micromolar affinity
for those on the 3-acetamide side chain.
Experimental Section
Table 3. TSPO binding affinity of the fluorescent probes.
General chemistry
Fluorescent probe
IC50 [μM]
Ki [nM]
Unless stated otherwise, all chemicals were laboratory or reagent
grade and were purchased from Merck (Australia) or AK Scientific,
Inc (USA). All chemicals were used as received. All solvents were
reagent grade and, when necessary, were purified and dried by
standard methods. Water was purified via Millipore filtration prior
to use. HOBt was purchased with added stabilizer (10% w/w H2O),
and therefore, the quantity required for reactions was adjusted
accordingly and is reflected in the reagent mass reported in the
experimental. All reactions requiring anhydrous conditions were
conducted under a positive atmosphere of nitrogen in oven-dried
glassware. Cold reaction temperatures were obtained by an ice
15a
15b
15c
15d
17
23a
23b
24
7.09�0.03
34.87�5.45
84.13�7.66
100.50�74.15
2.28�0.44
0.01�0.01
0.005�0.00
0.04�0.00
0.008�0.00
242.43�28.85
1205.57�334.28
2896.57�619.05
3276.30�2111.83
76.91�5.51
1.35�0.70
0.58�0.12
1.44�0.13
PK11195
0.59�0.20
The concentration of tested probes that inhibited [11C]PK11195 binding at
rat brain mitochondrial membranes (IC50) by 50% was determined with six
concentrations of the displacers, each performed in duplicate. The IC50
values were converted to an absolute constant Ki using the Cheng-Prusoff
equation.
°
bath (0 C). Heating of reaction was performed with a paraffin oil
bath. Standard syringe and autopipette techniques were used for
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the anhydrous addition of liquids. Reactions were routinely
monitored by TLC analysis performed on aluminium-backed SiO2
gel plates (F254 grade – 0.20 mm thickness) or by low resolution
mass spectrometry (LRMS) on Shimadzu LC-2010 mass spectrom-
eter. Visualization on TLC plates was achieved with UV light (λ=
254, 365 nm), ninhydrin stain, or cerium ammonium molybdate
stain. All filtrations were conducted as a gravity filtration through
a filter paper Whatman Grade 4 (20–25 μm) or as a vacuum
filtration through a sintered glass funnel (medium porosity).
Vacuum filtration was achieved with the aid of vacuum pump.
Organic solutions were dried over anhydrous Na2SO4. Solvent
removal via concentration was performed on a rotary evaporator
under reduced pressure. All solvent mixtures are expressed in
terms of volume ratio (i.e., v/v). Normal phase flash column
chromatography was performed on SiO2 gel 60 with a positive air
pressure. Preparative TLC was run using PLC silica gel 60 F254
1 mm plates (20×20 cm2). All synthesized compounds were dried
under high vacuum (<1 mbar) before determination of chemical
yields and spectroscopic characterization. All synthesized com-
pounds were subjected to full spectroscopic characterization and
assignment. Melting points were determined on a Gallenkamp
melting point apparatus in open capillary tubes and were
for 2 h. NaHCO3 (3.25 g, 38.69 mmol) was added and heating
continued for another 8 h. Additional NaHCO3 (2.25 g,
26.81 mmol) was added and stirring continued for a further
1
2
3
4
5
6
7
8
9
°
30 min. After cooling at 4 C for 18 h, the resulting solid was
collected by filtration, and washed with cold EtOH (50 mL), H2O
(100 mL), and EtOH (50 mL). The solid was then boiled with EtOH
(100 mL) for 15 min, cooled, filtered, and washed with EtOH
(50 mL) again and dried in vacuo to afford the imidazopyridine (6;
12.32 g, 73%) as an off-white solid. The spectroscopic data was in
agreement with that previously reported. TLC (CH2Cl2/MeOH
95:5): Rf =0.85. 1H NMR (400 MHz, DMSO): δ=8.78 (dd, J=2.0,
0.8 Hz, 1H), 8.26 (s, 1H), 7.89 (d, J=8.8 Hz, 2H), 7.59 (d, J=9.6 Hz,
1H), 7.26 (dd, J=9.6, 2.0 Hz), 7.01 (d, J=8.8 Hz, 2H), 3.81 (s, 3H).
13C NMR (400 MHz, DMSO): δ=159.2, 145.5, 143.2, 127.0, 126.0,
125.4, 124.6, 118.7, 117.1, 114.2, 108.7, 55.1. MS (ESI+ve) m/z 259
(35Cl [M+H]+, 100%), 261 (37Cl [M+H]+, 33%).
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1-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N,N-dimethylmethanamine (7)[34]
To a solution of imidazopyridine (6; 12.00 g, 46.35 mmol) in acetic
acid (145 mL) was slowly added an aqueous solution of dimeth-
ylamine (40% w/w – 74 mL, 0.58 mol), followed by aqueous
formaldehyde (37% w/w – 30 mL, 0.40 mol). The resulting mixture
1
uncorrected. H and 13C NMR spectra were recorded on a Bruker
Avance 400 (400 MHz) or Varian Inova 500 (500 MHz) NMR
spectrometer. Chemical shifts (δ) were reported in parts per
million (ppm) relative to the internal standard. Samples were
dissolved in CDCl3 (with TMS as the internal standard – 0.00 ppm),
CD3OD (solvent resonance as internal standard – 3.31 ppm) or [D6]
DMSO (solvent resonance as internal standard – 2.50 ppm). 13C
NMR signal assignments were confirmed by analysis of NMR
experiments: APT, gCOSY, gHSQC, gHMBC, zTOCSY, NOESY and/or
gHSQC-TOCSY. Low resolution mass spectrometry (LRMS) spectra
were recorded on a Shimadzu LC-2010 mass spectrometer in
electrospray positive and negative ionization modes (ESI-MS).
High resolution mass spectrometry (HRMS) spectra were recorded
on a Waters Quadrupole-Time of Flight (QTOF) Xevo or Thermo
LTQ Orbitrap XL 158 spectrometer in electrospray positive and
negative ionization modes (ESI-MS). All mass spectrometry
samples were dissolved in HPLC grade MeOH. Solid-state infrared
°
was stirred at 55 C for 18 h. After cooling to RT, the solution was
concentrated, and the syrupy residue was dissolved in a mixture
of H2O (60 mL) and CHCl3 (120 mL). Aqueous NaOH (10% w/v) was
added dropwise until the pH was 12. The organic layer was
separated, and the aqueous layer was further extracted with CHCl3
(2×30 mL). The combined organic layers were extracted with 2 N
HCl (3×60 mL) and the extract partially neutralized with NaOH
(8.70 g) to pH 3. After standing at RT for 18 h, the precipitated
hydroxymethyl by-product (1.03 g) was filtered. The cold filtrate
(ice bath) was basified to pH 12 with NaOH, and the resulting solid
was filtered, dried in vacuo and recrystallized from EtOH/H2O to
afford the tertiary amine (7; 10.05 g, 69%) as a pale-yellow
crystals. The spectroscopic data was in agreement with that
previously reported. TLC (CH2Cl2/MeOH 95:5): Rf =0.75. 1H NMR
(400 MHz, DMSO): δ=8.64 (dd, J=2.0, 0.8 Hz, 1H), 7.78 (d, J=
8.8 Hz, 2H), 7.62 (dd, J=9.6, 0.8 Hz, 1H), 7.31 (dd, J=9.6, 2.0 Hz,
1H), 7.04 (d, J=8.8 Hz, 2H), 3.89 (s, 2H), 3.80 (s, 3H), 2.16 (s, 6H).
13C NMR (400 MHz, DMSO): δ=159.0, 144.5, 142.3, 129.6, 126.5,
125.2, 123.5, 118.5, 117.8, 117.2, 113.9, 55.1, 51.4, 44.4. MS (ESI+
ve) m/z 316 (35Cl [M+H]+, 100%), 318 (37Cl [M+H]+, 33%).
spectroscopy was performed on
a Bruker Vertex 70 FTIR
spectrometer in combination with a MIRacle 10 Single Reflection
Attenuated Total Reflectance accessory outfitted with a 1.5 mm
round diamond crystal. IR peaks are reported as the wavenumber
(νmax in cmÀ 1) of the maximum absorption. Ultraviolet absorption
spectra were recorded on
a Schimadzu 1800 spectrometer.
Fluorescence spectra were recorded on a Cary Eclipse fluorescence
spectrophotometer with a slit width of 5 mm used for both
excitation and emission. Ultraviolet absorption and fluorescence
1-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N,N,N-trimethylmethanaminium iodide (8)[34]
°
spectra were recorded at ambient temperature (23�2 C). Com-
pounds were prepared as stock solutions in DMSO and diluted
such that the DMSO concentration did not exceed 1% v/v.
Analytical HPLC was performed on a Shimadzu LC-2030 C 3D UV/
Vis detector (λ=254 nm) using a biphenyl column Kinetex 100 Å
To a solution of the tertiary amine (7; 8.76 g, 27.83 mmol) in
toluene (110 mL) was added methyl iodide (6.0 mL, 96.38 mmol),
and the mixture was stirred at RT for 48 h in the dark. The
precipitated solid was filtered, washed with toluene (10 mL), and
dried in vacuo to afford the ammonium iodide (8; 10.66 g, 84%) as
a light-yellow solid. The product was used in the next reaction
step immediately or stored in the freezer for a limited time due to
stability issues. The spectroscopic data was in agreement with
that previously reported. TLC (CH2Cl2/MeOH 90:10): Rf =0.66. 1H
NMR (400 MHz, DMSO): δ=9.26 (br s, 1H), 7.79 (d, J=8.8 Hz, 2H),
7.75 (d, J=9.6 Hz, 1H), 7.50 (dd, J=9.6. 2.0 Hz, 1H), 7.06 (d, J=
8.8 Hz, 2H), 5.22 (s, 2H), 3.83 (s, 3H), 2.89 (s, 9H). 13C NMR
(400 MHz, DMSO): δ=159.5, 149.0, 144.2, 130.0, 127.3, 125.8,
123.3, 120.4, 118.0, 114.3, 109.8, 56.4, 55.2, 51.4. MS (ESI+ve) m/z
330 (35Cl [M], 100%), 332 (37Cl [M], 33%).
°
(150×4.60 mm; 5 μm) at 25 C using mobile phases A (water and
0.1% trifluoracetic acid (TFA)) and B (MeCN and 0.1% TFA) at a
flow rate of 1 mLminÀ 1. The following gradient was applied:
gradient elution for 35 min at 0–50% of solvent B, linear increase
from 0%–50% of solvent B over 25 min, hold at 50% of solvent B
for 10 min. The purity of all final compounds was found to be
higher than 95%.
6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine (6)[34]
To a solution of 2-bromo-4’-methoxyacetophenone (4; 15.00 g,
65.48 mmol) in EtOH (150 mL) was added 2-amino-5-chloropyr-
idine (3; 8.42 g, 65.48 mmol), and the reaction was stirred at reflux
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1810 (w), 1770 (w), 1683 (s), 1638 (s), 1539 (s), 1465 (s), 1390 (m),
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)
1
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5
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7
8
9
1285 (m), 1249 (s), 1174 (s), 1010 (s), 992 (s), 623 (s), 522 (s) cmÀ 1
.
acetamide (9)[34]
MS (ESI+ve) m/z 459 (35Cl [M+H]+, 100%), 461 (37Cl [M+H]+,
33%). HRMS (ESI+ve TOF) calcd for C23H2835ClN4O4 459.1794,
found 459.1792 ([M+H]+).
A solution of the ammonium iodide (8; 4.00 g, 8.74 mmol), KCN
(3.90 g, 59.89 mmol), and EtOH/H2O (1:1, 150 mL) was stirred at
reflux for 48 h. The solution was cooled to RT and evaporated to
dryness under a stream of N2. H2O (10 mL) was added and the
solid was filtered, washed with H2O (5 mL), and dried in vacuo to
afford the acetamide (9; 1.96 g, 71%) as a brown solid. The
spectroscopic data was in agreement with that previously
reported. TLC (CH2Cl2/MeOH 95:5): Rf =0.71. 1H NMR (400 MHz,
DMSO): δ=8.61 (d, J=2.0 Hz, 1H), 7.80 (br s, 1H), 7.73 (d, J=
8.8 Hz, 2H), 7.61 (d, J=9.6 Hz, 1H), 7.29 (dd, J=9.6, 2.0 Hz, 1H),
7.24 (br s, 1H), 7.05 (d, J=8.8 Hz, 2H), 3.98 (s, 2H), 3.81 (s, 3H). 13C
NMR (400 MHz, DMSO): δ=170.3, 159.0, 143.5, 142.2, 129.1, 126.5,
124.8, 122.9, 118.6, 117.2, 116.1, 114.1, 55.2, 30.5. MS (ESI+ve) m/z
316 (35Cl [M+H]+, 100%), 318 (37Cl [M+H]+, 33%).
Tert-butyl (3-(2-(6-chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)acetamido)propyl)carbamate (12b)
Following General Procedure A, acid (10; 300 mg, 0.95 mmol),
tert-butyl (3-aminopropyl)carbamate (11b; 196 mg, 1.14 mmol),
HOBt (141 mg, 0.95 mmol), EDCI (238 mg, 1.24 mmol), and DIPEA
(491 mg, 3.80 mmol) were stirred in anhydrous DMF (5.6 mL) for
24 h to afford the amide (12b; 364 mg, 81%) as a pale-yellow oil
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1
after flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.76. H
NMR (400 MHz, CDCl3): δ=8.16 (br s, 1H), 7.68 (d, J=8.8 Hz, 2H),
7.55 (d, J=9.6 Hz, 1H), 7.16 (dd, J=9.6, 2.0 Hz, 1H), 6.99 (d, J=
8.8 Hz, 2H), 6.92 (br s, 1H), 4.71 (t, J=5.2 Hz, 1H), 3.95 (s, 2H), 3.85
(s, 3H), 3.30–3.26 (m, 2H), 3.06–3.07 (m, 2H), 1.58–1.51 (m, 2H),
1.34 (s, 9H). 13C NMR (400 MHz, CDCl3): δ=168.4, 159.8, 156.9,
145.7, 143.7, 129.7, 126.4, 125.9, 121.6, 120.8, 117.9, 114.4, 114.0,
79.7, 55.5, 36.8, 35.9, 32.8, 30.3, 28.4. IR (neat)max 3344 (w), 3308
(w), 2980 (w), 2361 (w), 2340 (w), 1800 (w), 1776 (w), 1682 (s), 1644
(s), 1528 (s), 1457 (s), 1389 (m), 1364 (m), 1285 (m), 1246 (s), 1171
(s), 1027 (m), 787 (s), 626 (m), 531 (m) cmÀ 1. MS (ESI+ve) m/z 473
(35Cl [M+H]+, 100%), 475 (37Cl [M+H]+, 33%). HRMS (ESI+ve
TOF) calcd for C24H3035ClN4O4 473.1950, found 473.1947 ([M+H]+).
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)
acetic acid (10)[39]
To a solution of acetamide (9; 1.60 g, 5.07 mmol) in EtOH (35 mL)
was added a solution of KOH (2.42 g, 43.10 mmol) in H2O (6.0 mL).
The reaction was stirred at reflux for 18 h. The EtOH was removed
and the minimum volume of H2O was added to the residue to
dissolve completely any solid material. Acidification to pH 4 with
an aqueous HCl solution (12% v/v, 9 mL) precipitated the product
which was filtered, washed with H2O (20 mL), and dried in vacuo
to afford the acid (10; 1.51 g, 94%) as a grey solid. TLC (CH2Cl2/
MeOH 95:5): Rf =0.62. 1H NMR (400 MHz, DMSO): δ=9.24 (br s,
1H), 7.99–7.93 (m, 2H), 7.67 (d, J=8.8 Hz, 2H), 7.20 (d, J=8.8 Hz,
2H), 4.26 (s, 2H), 3.85 (s, 3H). 13C NMR (400 MHz, DMSO): δ=169.8,
160.7, 138.3, 135.2, 132.5, 129.8, 125.2, 123.2, 119.2, 116.8, 114.9,
113.4, 55.5, 29.4. MS (ESI+ve) m/z 317 (35Cl [M+H]+, 100%), 319
(37Cl [M+H]+, 33%).
Tert-butyl (4-(2-(6-chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)acetamido)butyl)carbamate (12c)
Following General Procedure A, acid (10; 300 mg, 0.95 mmol),
tert-butyl (4-aminobutyl)carbamate (11c; 215 mg, 1.14 mmol),
HOBt (141 mg, 0.95 mmol), EDCI (238 mg, 1.24 mmol), and DIPEA
(491 mg, 3.80 mmol) were stirred in anhydrous DMF (5.6 mL) for
24 h to afford the amide (12c; 420 mg, 91%) as a pale-yellow oil
1
after flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.71. H
General Procedure A: Amide coupling
NMR (400 MHz, DMSO): δ=8.61 (br s, 1H), 8.32 (t, J=5.6 Hz, 1H),
7.72 (d, J=8.8 Hz, 2H), 7.61 (d, J=9.6 Hz, 1H), 7.29 (dd, J=9.6,
2.0 Hz, 1H), 7.03 (d, J=8.8 Hz, 2H), 6.78 (t, J=5.6 Hz, 1H), 3.98 (s,
2H), 3.81 (s, 3H), 3.13–3.08 (m, 2H), 2.96–2.88 (m, 2H), 1.45–1.37
(m, 4H), 1.36 (s, 9H). 13C NMR (400 MHz, DMSO): δ=168.0, 159.0,
155.6, 143.6, 142.2, 129.1, 126.5, 124.8, 122.9, 118.6, 117.2, 115.9,
114.0, 77.3, 55.1, 40.2, 38.6, 30.7, 28.2, 27.0, 26.4. IR (neat)max 3310
(w), 3256 (w), 2935 (w), 1790 (w), 1780 (w), 1682 (m), 1639 (m),
1560 (m), 1535 (m), 1500 (m), 1365 (m), 1285 (m), 1249 (s), 1174
(m), 1025 (s), 990 (s), 623 (s), 522 (s) cmÀ 1. MS (ESI+ve) m/z 487
(35Cl [M+H]+, 100%), 489 (37Cl [M+H]+, 33%). HRMS (ESI+ve
TOF) calcd for C25H3235ClN4O4 487.2107, found 487.2105 ([M+H]+).
The acid (10; 1.0 equiv.), the appropriate mono-Boc-protected
polymethylendiamine (11a–d; 1.2 equiv.), HOBt (1.0 equiv.), EDCI
(1.3 equiv.), and DIPEA (4.0 equiv.) were combined in anhydrous
DMF (6.0 mL/mmol acid), and stirred at RT for 24 h under N2. The
resulting reaction mixture was poured into H2O (25 mL) and
extracted with EtOAc (3×25 mL). The organic layer was washed
with H2O (2×25 mL), brine (2×25 mL), dried (Na2SO4), filtered, and
concentrated. The residue was subjected to flash chromatography
over SiO2 gel (CH2Cl2/MeOH 95:5) to afford the desired amides
12a–d.
Tert-butyl (2-(2-(6-chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)acetamido)ethyl)carbamate (12a)
Tert-butyl (6-(2-(6-chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)acetamido)hexyl)carbamate (12d)
Following General Procedure A, acid (10; 220 mg, 0.69 mmol),
tert-butyl (2-aminoethyl)carbamate (11a; 133 mg, 0.83 mmol),
HOBt (103 mg, 0.69 mmol), EDCI (173 mg, 0.90 mmol), and DIPEA
(357 mg, 2.76 mmol) were stirred in anhydrous DMF (4.1 mL) for
24 h to afford the amide (12a; 233 mg, 74%) as a pale-yellow oil
Following General Procedure A, acid (10; 300 mg, 0.95 mmol),
tert-butyl (6-aminohexyl)carbamate (11d; 247 mg, 1.14 mmol),
HOBt (141 mg, 0.95 mmol), EDCI (238 mg, 1.24 mmol), and DIPEA
(491 mg, 3.80 mmol) were stirred in anhydrous DMF (5.6 mL) for
24 h to afford the amide (12d; 470 mg, 96%) a pale-yellow oil
1
after flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.78. H
NMR (400 MHz, CDCl3): δ=8.12 (br s, 1H), 7.64 (d, J=8.8 Hz, 2H),
7.53 (d, J=9.6 Hz, 1H), 7.16 (dd, J=9.6, 2.0 Hz, 1H), 6.99 (d, J=
8.8 Hz, 2H), 6.71 (br s, 1H), 4.83 (br s, 1H), 3.93 (s, 2H), 3.85 (s, 3H),
3.39–3.35 (m, 2H), 3.22–3.18 (m, 2H), 1.36 (s, 9H). 13C NMR
(400 MHz, CDCl3): δ=168.7, 159.9, 157.2, 145.8, 143.7, 129.7, 126.2,
126.1, 121.6, 120.9, 117.8, 114.5, 113.7, 80.1, 55.5, 41.5, 40.5, 32.7,
28.4. IR (neat)max 3310 (m), 3256 (m), 2935 (w), 2361 (w), 2339 (w),
1
after flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.70. H
NMR (400 MHz, DMSO): δ=8.60 (br s, 1H), 8.32 (t, J=5.6 Hz, 1H),
7.74 (d, J=8.8 Hz, 2H), 7.62 (d, J=9.6 Hz, 1H), 7.31 (dd, J=9.6,
2.0 Hz, 1H), 7.04 (d, J=8.8 Hz, 2H), 6.74 (t, J=5.6 Hz, 1H), 3.98 (s,
2H), 3.81 (s, 3H), 3.13–3.08 (m, 2H), 2.91–2.87 (m, 2H), 1.46–1.21
(m, 8H), 1.36 (s, 9H). 13C NMR (400 MHz, DMSO): δ=168.1, 159.1,
155.7, 143.6, 142.3, 129.3, 126.5, 125.0, 122.9, 118.8, 117.3, 116.0,
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114.1, 77.5, 55.2, 40.0, 39.0, 30.8, 29.6, 29.1, 28.4, 26.2, 26.1. IR
(neat)max 3292 (w), 2931 (w), 1800 (w), 1776 (w), 1688 (s), 1651 (s),
1500 (s), 1456 (m), 1390 (s), 1364 (s), 1329 (m), 1248 (s), 1172 (s),
1026 (s), 837 (s), 799 (s), 626 (s) cmÀ 1. MS (ESI+ve) m/z 515 (35Cl
[M+H]+, 100%), 517 (37Cl [M+H]+, 33%). HRMS (ESI+ve TOF)
calcd for C27H3635ClN4O4 515.2420, found 515.2429 ([M+H]+).
CF3CO2H (1.2 mL) to afford the free amine (13c; 85%) as a pale-
yellow foam that collapsed into a translucent gum after flash
chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.46. 1H NMR
(400 MHz, DMSO): δ=8.87 (br s, 1H), 8.47 (t, J=5.6 Hz, 1H), 7.90–
7.80 (m, 3H), 7.70 (d, J=8.8 Hz, 2H), 7.63 (d, J=9.2 Hz, 1H), 7.12 (d,
J=8.8 Hz, 2H), 4.05 (s, 2H), 3.84 (s, 3H), 3.15–3.11 (m, 2H), 2.85–
2.76 (m, 2H), 1.60–1.46 (m, 4H). 13C NMR (400 MHz, DMSO): δ=
167.5, 161.1, 138.5, 134.9, 133.1, 130.4, 125.8, 123.7, 119.5, 118.3,
115.3, 113.6, 60.0, 38.7, 38.7, 30.4, 26.4, 24.8. IR (neat)max 3300 (w),
3161 (w), 2960 (w), 1777 (w), 1640 (m), 1611 (m), 1555 (m), 1510
(m), 1260 (m), 1180 (s), 1130 (s), 1023 (m), 906 (m), 820 (s), 796 (s),
704 (s), 518 (s) cmÀ 1. MS (ESI+ve) m/z 387 (35Cl [M+H]+, 100%),
389 (37Cl [M+H]+, 33%). HRMS (ESI+ve TOF) calcd for
C20H2435ClN4O2 387.1582, found 387.1583 ([M+H]+).
1
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4
5
6
7
8
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General Procedure B: Amine deprotection
The N-protected amine (12a–d; 1.0 equiv.) in CH2Cl2 (10 mL/mmol
amine) was treated with CF3CO2H (3 mL/mmol amine). The
reaction mixture was stirred at RT for 4 h followed by removal of
the solvent. The residue was dissolved in EtOAc (25 mL),
neutralized with saturated aqueous NaHCO3, and then washed
with H2O (2×25 mL). The organic layer was dried (Na2SO4) and
concentrated. The residue was subjected to flash chromatography
over SiO2 gel (CH2Cl2/MeOH 95:5) to afford the desired free
amines 13a–d.
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N-(6-Aminohexyl)-2-(6-chloro-2-(4-methoxyphenyl)imidazo
[1,2-a]pyridin-3-yl)acetamide (13d)
Following General Procedure B, N-protected amine (12d; 225 mg,
0.44 mmol) was dissolved in CHCl2 (4.4 mL) and treated with
CF3CO2H (1.3 mL) to afford the free amine (13d; 160 mg, 87%) as
a pale-yellow foam that collapsed into a translucent gum after
N-(2-Aminoethyl)-2-(6-chloro-2-(4-methoxyphenyl)imidazo
[1,2-a]pyridin-3-yl)acetamide (13a)
1
flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.45. H NMR
Following General Procedure B, N-protected amine (12a; 110 mg,
0.24 mmol) was dissolved in CHCl2 (2.4 mL) and treated with
CF3CO2H (0.7 mL) to afford the free amine (13a; 75 mg, 87%) as a
pale-yellow foam that collapsed into a translucent gum after flash
chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.50. 1H NMR
(400 MHz, DMSO): δ=8.59 (br s, 1H), 8.49 (t, J=5.6 Hz, 1H), 7.68
(d, J=8.8 Hz, 2H), 7.61 (d, J=9.2 Hz, 1H), 7.31 (dd, J=9.2, 2.0 Hz,
1H), 7.04 (d, J=8.8 Hz, 2H), 4.03 (s, 2H), 3.80 (s, 3H), 3.47 (br s, 2H),
3.38 (t, J=6.0 Hz, 2H), 2.92 (t, J=6.0 Hz, 2H’). 13C NMR (400 MHz,
DMSO): δ=169.3, 159.1, 143.8, 142.4, 129.3, 126.5, 125.2, 123.1,
118.8, 117.3, 115.6, 114.2, 55.3, 38.9, 36.9, 30.8. IR (neat)max 3336
(w), 3085 (w), 2944 (w), 1779 (m), 1661 (m), 1563 (m), 1509 (m)
1260 (m), 1224 (s), 1140 (s), 1026 (m), 834 (s), 809 (s), 704 (s), 517
(s) cmÀ 1. MS (ESI+ve) m/z 359 (35Cl [M+H]+, 100%), 361 (37Cl [M+
H]+, 33%). HRMS (ESI+ve TOF) calcd for C18H2035ClN4O2 359.1269,
found 359.1263 ([M+H]+).
(400 MHz, DMSO): δ=9.22 (br s, 1H), 8.68 (t, J=5.6 Hz, 1H), 8.05
(br s, 2H), 7.99–7.93 (m, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.16 (d, J=
8.8 Hz, 2H), 4.14 (s, 2H), 3.85 (s, 3H), 3.14–3.09 (m, 2H), 2.77–2.69
(m, 2H), 1.58–1.24 (m, 8H). 13C NMR (400 MHz, DMSO): δ=167.3,
161.1, 138.8, 135.6, 132.6, 130.4, 125.6, 123.5, 120.0, 118.3, 115.2,
113.9, 56.0, 30.4, 29.2, 27.3, 27.2, 26.4, 26.0, 25.8. IR (neat)max 3337
(w), 3086 (w), 2943 (w), 1779 (m), 1661 (m), 1609 (m), 1563 (m),
1510 (m), 1240 (m), 1139 (s), 1026 (s), 908 (m), 834 (s), 784 (s), 704
(s), 516 (s) cmÀ 1. MS (ESI+ve) m/z 415 (35Cl [M+H]+, 100%), 417
(37Cl [M+H]+, 33%). HRMS (ESI+ve TOF) calcd for C22H2835ClN4O2
415.1895, found 415.1895 ([M+H]+).
General Procedure C: Synthesis of fluorescent probes
The amine (13a–d; 1.0 equiv.) was dissolved in anhydrous DMF
(20 mL/mmol amine) with magnetic stirring. The solution was
°
brought to 0 C (ice bath) followed by sequential dropwise
N-(3-Aminopropyl)-2-(6-chloro-2-(4-methoxyphenyl)imidazo
[1,2-a]pyridin-3-yl)acetamide (13b)
addition of DIPEA (1.4 equiv.) and a solution of NBDÀ Cl (14;
1.0 equiv.) in DMF (1.0 mL) under a flow of N2 gas. The reaction
vessel was then sealed and allowed to warm to RT and stirring
was continued for 18 h under N2 in the dark. The solution was
partitioned between CHCl3 (100 mL) and H2O (100 mL). The
aqueous phase was separated and extracted with CHCl3 (5×
50 mL). The combined organic extracts were washed with H2O (2×
100 mL), brine (2×100 mL), dried (Na2SO4), filtered, and concen-
trated. The residue was purified by preparative TLC plate
chromatography to afford the desired fluorescent probes 15a–d.
Following General Procedure B, N-protected amine (12b; 200 mg,
0.42 mmol) was dissolved in CHCl2 (4.2 mL) and treated with
CF3CO2H (1.3 mL) to afford the free amine (13b; 140 mg, 89%) as
a pale-yellow foam that collapsed into a translucent gum after
1
flash chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.49. H NMR
(400 MHz, DMSO): δ=8.89 (br s, 1H), 8.54 (t, J=5.6 Hz, 1H), 7.89–
7.79 (m, 3H), 7.70 (d, J=8.8 Hz, 2H), 7.64 (d, J=9.2 Hz, 1H), 7.12 (d,
J=8.8 Hz, 2H), 4.07 (s, 2H), 3.83 (s, 3H), 3.26–3.23 (m, 2H), 2.86–
2.76 (m, 2H), 1.76 (quint, J=6.0 Hz, 2H). 13C NMR (400 MHz,
DMSO): δ=167.7, 161.2, 138.6, 135.1, 133.1, 130.4, 125.8, 123.8,
119.5, 118.2, 115.4, 113.6, 56.0, 37.1, 36.6, 30.4, 27.5. IR (neat)max
3370 (w), 2962 (w), 2728 (w), 1656 (s), 1508 (m), 1420 (m), 1354
(m), 1301 (m), 1255 (m), 1174 (s), 1125 (s), 1019 (m), 833 (s), 795
(s), 720 (s), 518 (s) cmÀ 1. MS (ESI+ve) m/z 373 (35Cl [M+H]+,
100%), 375 (37Cl [M+H]+, 33%). HRMS (ESI+ve TOF) calcd for
C19H2235ClN4O2 373.1426, found 373.1424 ([M+H]+).
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)
ethyl)acetamide (15a)
Following General Procedure C, amine (13a; 40 mg, 0.11 mmol)
was dissolved in DMF (1.1 mL) followed by sequential addition of
DIPEA (19 mg, 0.15 mmol) and NBDÀ Cl (14; 22 mg, 0.11 mmol) to
afford the desired fluorescent probe (15a; 31 mg, 54%) as a light-
brown solid after preparative TLC plate chromatography (CH2Cl2/
N-(4-Aminobutyl)-2-(6-chloro-2-(4-methoxyphenyl)imidazo
[1,2-a]pyridin-3-yl)acetamide (13c)
°
MeOH 95:5). TLC (CH2Cl2/MeOH 95:5): Rf =0.33. mp 206–208 C.
1H NMR (500 MHz, DMSO): δ=9.31 (br s, 1H), 8.54–8.48 (m, 2H),
8.31 (d, J=8.5 Hz, 1H), 7.71 (d, J=8.5 Hz, 2H), 7.57 (d, J=9.5 Hz,
1H), 7.20 (d, J=9.5 Hz, 1H), 6.92 (d, J=8.5 Hz, 2H), 6.31 (d, J=
8.5 Hz, 1H), 3.97 (s, 2H), 3.77 (s, 3H), 3.60 (br s, 2H), 3.50–3.45 (m,
Following General Procedure B, N-protected amine (12c; 200 mg,
0.41 mmol) was dissolved in CHCl2 (4.1 mL) and treated with
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2H). 13C NMR (500 MHz, DMSO): δ=169.4, 159.4, 145.9, 144.8,
144.5, 144.1, 142.7, 138.2, 129.5, 126.8, 125.3, 123.1, 121.4, 119.1,
117.6, 115.7, 114.3, 99.3, 55.5, 43.2, 38.4, 31.3. IR (neat)max 3363 (w),
3276 (w), 3100 (w), 1760 (w), 1654 (m), 1632 (m), 1589 (s), 1539 (s),
1517 (s), 1303 (s), 1137 (s), 1022 (m), 999 (s), 828 (s), 778 (s), 738
(s), 623 (s) cmÀ 1. MS (ESIÀ ve) m/z 520 (35Cl [MÀ H]À , 100%), 522
(37Cl [MÀ H]À , 33%). HRMS (ESIÀ ve TOF) calcd for C24H1935ClN7O5
520.1142, found 520.1136 ([MÀ H]À ).
brown solid after preparative TLC plate chromatography (CH2Cl2/
1
2
3
4
5
6
7
8
9
°
MeOH 90:10). TLC (CH2Cl2/MeOH 90:10): Rf =0.30. mp 210–211 C.
1H NMR (400 MHz, DMSO): δ=9.53 (t, J=4.4 Hz, 1H), 8.60 (s, 1H),
8.48 (d, J=9.2 Hz, 1H), 8.32 (t, J=4.4 Hz, 1H), 7.73 (d, J=8.8 Hz,
2H), 7.60 (d, J=9.6 Hz, 1H), 7.27 (dd, J=9.6, 2.0 Hz, 1H), 7.00 (d,
J=8.8 Hz, 2H), 6.36 (d, J=9.2 Hz, 1H), 3.98 (s, 2H), 3.78 (s, 3H), 3.33
(br s, 2H), 3.15–3.10 (m, 2H), 1.69 (quint, J=6.4 Hz, 2H), 1.48 (quint,
J=6.4 Hz, 2H), 1.46–1.35 (m, 4H). 13C NMR (400 MHz, DMSO): δ=
168.0, 158.9, 145.1, 144.4, 144.1, 143.5, 142.1, 137.9, 129.1, 126.4,
124.9, 122.8, 120.5, 118.6, 117.2, 115.9, 113.9, 99.0, 55.1, 43.3, 38.7,
30.7, 28.9, 27.6, 26.1, 26.0. IR (neat)max 3255 (w), 3225 (w), 2922 (w),
2852 (w), 1640 (w), 1650 (w), 1615 (m), 1527 (m), 1496 (m), 1442
(m), 1292 (s), 1245 (s), 1173 (s), 1025 (m), 998 (m), 903 (m), 836
(m), 801 (m), 739 (m), 595 (s), 520 (s) cmÀ 1. MS (ESI+ve) m/z 578
(35Cl [M+H]+, 100%), 580 (37Cl [M+H]+, 33%). HRMS (ESI+ve
TOF) calcd for C28H2935ClN7O5 578.1913, found 578.1911 ([M+H]+).
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)
propyl)acetamide (15b)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Following General Procedure C, amine (13b; 150 mg, 0.40 mmol)
was dissolved in DMF (3.0 mL) followed by sequential addition of
DIPEA (72 mg, 0.56 mmol) and NBDÀ Cl (14; 80 mg, 0.40 mmol) to
afford the desired fluorescent probe (15b; 90 mg, 42%) as a light-
brown solid after preparative TLC plate chromatography (CH2Cl2/
7-Nitrobenzo[c][1,2,5]oxadiazol-4-amine (16)[40]
°
MeOH 90:10). TLC (CH2Cl2/MeOH 90:10): Rf =0.48. mp 207–209 C.
1H NMR (400 MHz, DMSO): δ=9.47 (t, J=4.4 Hz, 1H), 8.64 (br s,
1H), 8.47 (d, J=9.2 Hz, 1H), 8.38 (t, J=4.4 Hz, 1H), 7.73 (d, J=
8.8 Hz, 2H), 7.62 (d, J=9.6 Hz, 1H), 7.29 (dd, J=9.6, 2.0 Hz, 1H),
7.01 (d, J=8.8 Hz, 2H), 6.34 (d, J=9.2 Hz, 1H), 4.02 (s, 2H), 3.78 (s,
3H), 3.49 (br s, 2H), 3.28–3.23 (m, 2H), 1.92–1.85 (m, 2H). 13C NMR
(400 MHz, DMSO): δ=168.8, 159.5, 145.6, 144.9, 144.6, 143.9,
142.6, 138.3, 129.6, 126.8, 125.5, 123.4, 121.3, 119.2, 117.6, 116.2,
114.5, 99.5, 55.6, 41.6, 37.0, 31.2, 28.1. IR (neat)max 3231 (w), 3072
(w), 2966 (w), 1717 (w), 1633 (m), 1582 (m), 1523 (m), 1495 (m),
1446 (m), 1293 (s), 1249 (s), 1175 (s), 1137 (s), 1010 (m), 802 (s),
597 (s), 526 (s) cmÀ 1. MS (ESIÀ ve) m/z 534 (35Cl [MÀ H]À , 100%),
536 (37Cl [MÀ H]À , 33%). HRMS (ESIÀ ve TOF) calcd for
C25H2135ClN7O5 534.1298, found 534.1305 ([MÀ H]À ).
To a stirred solution of 4-chloro-7-nitrobenzofurazan (14; 500 mg,
°
2.51 mmol) in MeOH (25 mL) at 0 C (ice bath) was added dropwise
NH4OH (30% v/v – 10 mL, 1.48 mol). The reaction was allowed to
warm to RT and stirring was continued for 24 h under N2 in the
dark. The solvent was evaporated in vacuo, and the residue was
purified by flash chromatography over SiO2 gel (EtOAc/hexanes
70:30) to afford the NBD amine (16; 229 mg, 51%) as a dark-
brown solid. The spectroscopic data was in agreement with that
1
previously reported. TLC (EtOAc/hexanes 70:30): Rf =0.50. H NMR
(400 MHz, DMSO): δ=8.83 (br s, 2H), 8.45 (d, J=8.8 Hz, 1H), 6.37
(d, J=8.8 Hz, 1H). 13C NMR (400 MHz, DMSO): δ=147.7, 144.6,
144.4, 138.3, 120.8, 103.1. MS (ESIÀ ve) m/z 179 ([MÀ H]À , 100%).
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)acetamide
(17)
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N-(4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)
butyl)acetamide (15c)
To a stirred solution of the acid (10; 33 mg, 0.10 mmol) in
anhydrous DMF (2.0 mL) was added HOBt (15 mg, 0.10 mmol),
EDCI (25 mg, 0.13 mmol), DIPEA (52 mg, 0.40 mmol), and the NBD-
NH2 (16; 20 mg, 0.10 mmol). Stirring was prolonged at RT for 48 h,
and the resulting reaction mixture was poured into H2O (25 mL)
and extracted with EtOAc (3×25 mL). The organic solution was
separated, washed with H2O (2×25 mL), brine (2×25 mL), dried
(Na2SO4), filtered, and concentrated. The residue was subjected to
flash chromatography over SiO2 gel (CH2Cl2/MeOH 95:5) to afford
Following General Procedure C, amine (13c; 100 mg, 0.26 mmol)
was dissolved in DMF (1.6 mL) followed by sequential addition of
DIPEA (47 mg, 0.36 mmol) and NBDÀ Cl (14; 52 mg, 0.26 mmol) to
afford the desired fluorescent probe (15c; 82 mg, 57%) as a light-
brown solid after preparative TLC plate chromatography (CH2Cl2/
°
MeOH 93:7). TLC (CH2Cl2/MeOH 93:7): Rf =0.31. mp 208–209 C.
1H NMR (500 MHz, DMSO): δ=9.55 (t, J=4.4 Hz, 1H), 8.61 (s, 1H),
8.40 (d, J=9.2 Hz, 1H), 8.36 (t, J=4.4 Hz, 1H), 7.72 (d, J=8.8 Hz,
2H), 7.61 (d, J=9.6 Hz, 1H), 7.27 (d, J=9.6 Hz, 1H), 6.99 (d, J=
8.8 Hz, 2H), 6.38 (d, J=9.2 Hz, 1H), 3.97 (s, 2H), 3.77 (s, 3H), 3.46 (br
s, 2H), 3.22–3.16 (m, 2H), 1.74–1.66 (m, 2H), 1.61–1.53 (m, 2H). 13C
NMR (500 MHz, DMSO): δ=168.6, 159.4, 145.6, 144.9, 142.6, 138.4,
133.0, 131.0, 129.6, 126.8, 125.4, 123.4, 121.1, 119.1, 117.6, 116.4,
114.4, 99.5, 55.6, 44.4, 38.8, 31.2, 27.0, 25.5. IR (neat)max 3368 (w),
3218 (w), 3081 (w), 2914 (w), 1735 (w), 1661 (m), 1620 (m), 1564
(m), 1506 (m), 1461 (m), 1316 (s), 1231 (s), 1172 (s), 1017 (s), 908
(m), 829 (s), 807 (s), 741 (s), 599 (s), 523 (s) cmÀ 1. MS (ESI+ve) m/z
550 (35Cl [M+H]+, 100%), 552 (37Cl [M+H]+, 33%). HRMS (ESI+ve
TOF) calcd for C26H2535ClN7O5 550.1600, found 550.1601 ([M+H]+).
the desired fluorescent probe (17; 30 mg, 62%) as a light-brown
1
°
solid. TLC (CH2Cl2/MeOH 95:5): Rf =0.63. mp 167–169 C. H NMR
(500 MHz, CD3OD): δ=8.97 (br s, 1H), 8.47 (d, J=8.0 Hz, 1H), 7.92–
7.87 (m, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.64 (d, J=9.0 Hz, 2H), 7.16 (d,
J=9.0 Hz, 2H), 6.40 (d, J=8.0 Hz, 1H), 4.22 (s, 2H), 3.89 (s, 3H). 13C
NMR (500 MHz, CD3OD): δ=171.7, 163.0, 148.5, 148.2, 145.6, 145.6,
140.3, 138.4, 138.1, 134.0, 131.2, 126.2, 125.8, 120.6, 118.3, 116.1,
114.3, 103.2, 56.1, 30.1. IR (neat)max 3335 (w), 2930 (w), 1700 (w),
1647 (w), 1558 (m), 1539 (s), 1507 (m), 1251 (s), 1174 (s), 1024 (s),
992 (s), 796 (s), 732 (s), 612 (s) cmÀ 1. MS (ESIÀ ve) m/z 477 (35Cl
[MÀ H]À , 100%), 479 (37Cl [MÀ H]À , 33%). HRMS (ESIÀ ve TOF) calcd
for C22H1435ClN6O5 477.0720, found 477.0714 ([MÀ H]À ).
2-(6-Chloro-2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)
hexyl)acetamide (15d)
2-(6-Chloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)
acetic acid hydrobromide hydrate (18)[34]
Acetamide (9; 1.60 g, 5.07 mmol) was dissolved in acetic acid
(12.5 mL, 0.21 mol) with magnetic stirring, and the solution was
Following General Procedure C, amine (13d; 100 mg, 0.24 mmol)
was dissolved in DMF (1.4 mL) followed by sequential addition of
DIPEA (44 mg, 0.34 mmol) and NBDÀ Cl (14; 48 mg, 0.24 mmol) to
afford the desired fluorescent probe (15d; 78 mg, 56%) as a light-
°
brought to 0 C (ice bath). HBr (48% w/w – 25 mL, 0.11 mol) was
added dropwise, the ice bath removed, and the reaction solution
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°
heated to 110 C for 48 h. The solution was cooled to RT, then
3.36–3.29 (m, 4H), 1.39 (s, 9H), 1.16 (t, J=7.2 Hz, 3H), 1.07 (t, J=
7.2 Hz, 3H). 13C NMR (400 MHz, DMSO): δ=167.0, 158.2, 155.7,
143.4, 142.2, 129.0, 126.7, 124.7, 123.0, 118.4, 117.1, 116.4, 114.6,
77.8, 66.5, 63.6, 41.7, 38.3, 28.7, 28.2, 14.1, 13.0. IR (neat)max 3458
(w), 3273 (w), 2975 (w), 1703 (m), 1638 (m), 1500 (m), 1390 (w),
1245 (m), 1171 (m), 1035 (s), 1025 (s), 1010 (s), 820 (m), 758 (m),
525 (m) cmÀ 1. MS (ESI+ve) m/z 501 (35Cl [M+H]+, 100%), 503 (37Cl
[M+H]+, 33%). HRMS (ESI+ve TOF) calcd for C26H3435ClN4O4
501.2263, found 501.2269 ([M+H]+).
1
2
3
4
5
6
7
8
9
°
placed at 4 C for 18 h to give a brown precipitate. The precipitate
was filtered, washed with acetic acid (10 mL), and dried in vacuo
to afford the acid (18; 1.22 g, 63%) as a light-brown solid. The
spectroscopic data was in agreement with that previously
reported. TLC (CH2Cl2/MeOH 90:5): Rf =0.58. 1H NMR (400 MHz,
DMSO): δ=10.16 (br s, 1H), 9.28 (s, 1H), 8.03–7.96 (m, 2H), 7.52 (d,
J=8.8 Hz, 2H), 7.02 (d, J=8.8 Hz, 2H), 4.24 (s, 2H). 13C NMR
(400 MHz, DMSO): δ=169.8, 159.4, 137.9, 135.1, 132.8, 129.9,
125.4, 123.5, 117.1, 116.7, 116.3, 113.2, 29.3. MS (ESI+ve) m/z 303
(35Cl [M+H]+, 100%), 305 (37Cl [M+H]+, 33%).
Tert-butyl (3-(4-(6-chloro-3-(2-(diethylamino)-2-oxoethyl)
imidazo[1,2-a]pyridin-2-yl)phenoxy)propyl)carbamate (21b)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
2-(6-Chloro-2-(4-hydroxyphenyl)imidazo[1,2-a]
pyridin-3-yl)-N,N-diethylacetamide (19)[34]
Following General Procedure D, phenol (19; 250 mg, 0.70 mmol)
was dissolved in anhydrous DMF (10.5 mL) followed by sequential
addition of K2CO3 (387 mg, 28 mmol) and tert-butyl (3-bromoprop-
yl)carbamate (20b; 200 mg, 0.84 mmol) to afford the O-alkylated
phenol (21b; 261 mg, 72%) as a pale-yellow foam after prepara-
tive TLC plate chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.76.
NMR (400 MHz, DMSO): δ=8.51 (br s, 1H), 7.60 (d, J=9.6 Hz, 1H),
7.52 (d, J=8.8 Hz, 2H), 7.27 (dd, J=9.6, 2.0 Hz, 1H), 7.01 (d, J=
8.8 Hz, 2H), 6.90 (t, J=5.2 Hz, 1H), 4.20 (s, 2H), 4.02 (t, J=6.4 Hz,
2H), 3.44 (q, J=7.2 Hz, 2H), 3.33 (q, J=7.2 Hz, 2H), 3.12–3.08 (m,
2H), 1.85 (quint, J=6.4 Hz, 2H), 1.38 (s, 9H), 1.17 (t, J=7.2 Hz, 3H),
1.07 (t, J=7.2 Hz, 3H). 13C NMR (400 MHz, DMSO): δ=167.0, 158.3,
155.6, 143.4, 142.2, 129.0, 126.5, 124.7, 123.0, 118.4, 117.1, 116.4,
114.5, 77.5, 65.3, 63.6, 41.7, 36.9, 29.2, 28.7, 28.2, 14.1, 13.0. IR
(neat)max 3394 (w), 3250 (w), 2974 (w), 1633 (m), 1501 (m), 1391
(w), 1332 (w), 1248 (m), 1048 (m), 1037 (s), 998 (s), 823 (m), 761
(m), 525 (m) cmÀ 1. MS (ESI+ve) m/z 515 (35Cl [M+H]+, 100%), 517
(37Cl [M+H]+, 33%). HRMS (ESI+ve TOF) calcd for C27H3635ClN4O4
515.2420, found 515.2426 ([M+H]+).
A
mixture of acid (18; 830 mg, 2.16 mmol), HOBt (295 mg,
2.16 mmol), EDCI (539 mg, 2.81 mmol), diethylamine (268 μL,
2.59 mmol), and DIPEA (1.58 mL, 8.64 mmol) was stirred in
anhydrous DMF (8 mL) at RT for 24 h under N2. H2O (43 mL) and
acetic acid (0.4 mL) were added, stirred for 5 min, and placed at
4 C for 18 h to give a brown precipitate which was filtered,
washed with H2O (20 mL), and dried in vacuo. Anhydrous DMF
(4.0 mL) and diethylamine (0.4 mL) were added to the crude
product and heated to 110 C for 6 h until the diethylamine was
evaporated from the solution. After cooling to 80 C, EtOAc
(8.0 mL) was added rapidly. The solution was cooled to 4 C for
18 h to form a white crystalline solid, which was filtered, washed
with cold ethyl acetate (4.0 mL), and dried in vacuo to afford
diethylamide (19; 570 mg, 74%) as white needles. The spectro-
scopic data was in agreement with that previously reported. TLC
°
°
°
°
1
(CH2Cl2/MeOH 95:5): Rf =0.67. H NMR (400 MHz, DMSO): δ=9.62
(br s, 1H), 8.50 (dd, J=0.8, 2.0 Hz, 1H), 7.59 (dd, J=9.6, 0.8 Hz, 1H),
7.42 (d, J=8.8 Hz, 2H), 7.27 (dd, J=9.6, 2.0 Hz, 1H), 6.84 (d, J=
8.8 Hz, 2H), 4.18 (s, 2H), 3.44 (q, J=7.2 Hz, 2H), 3.32 (q, J=7.2 Hz,
2H), 1.16 (t, J=7.2 Hz, 3H), 1.06 (t, J=7.2 Hz, 3H). 13C NMR
(400 MHz, DMSO): δ=167.1, 157.2, 143.9, 142.1, 129.1, 125.0,
124.5, 123.0, 118.3, 117.0, 116.1, 115.4„ 41.7, 39.9, 28.7, 14.1, 13.1.
MS (ESI+ve) m/z 358 (35Cl [M+H]+, 100%), 100%), 360 (37Cl [M+
H]+, 33%).
General Procedure E: Amine deprotection
The O-alkylated phenol (21a–b; 1.0 equiv.) in CH2Cl2 (10 mL/mmol
amine) was treated with CF3CO2H (3 mL/mmol amine). The
reaction mixture was stirred at RT for 4 h followed by removal of
the solvent. The residue was dissolved in EtOAc (25 mL),
neutralized with saturated aqueous NaHCO3, and then washed
with H2O (2×25 mL). The organic layer was dried (Na2SO4) and
concentrated. The residue was purified by preparative TLC plate
chromatography (CH2Cl2/MeOH 95:5) to afford the desired free
amines 22a–b.
General Procedure D: Phenol alkylation
To a stirred solution of the phenol (19; 1.0 equiv.) in anhydrous
DMF (15 mL/mmol phenol) was added K2CO3 (4.0 equiv.) and the
appropriate Boc-protected bromoamine (20a–b; 1.2 equiv.). Stir-
ring was prolonged at RT for 48 h under N2, and the resulting
reaction mixture was poured into H2O (25 mL) and extracted with
EtOAc (3×25 mL). The organic solution was separated, washed
with H2O (2×25 mL), brine (2×25 mL), dried (Na2SO4), filtered, and
concentrated. The residue was purified by preparative TLC plate
chromatography (CH2Cl2/MeOH 95:5) to afford the desired O-
alkylated phenols 21a–b.
2-(2-(4-(2-Aminoethoxy)phenyl)-6-chloroimidazo[1,2-a]
pyridin-3-yl)-N,N-diethylacetamide (22a)
Following General Procedure E, O-alkylated phenol (21a; 200 mg,
0.40 mmol) was dissolved in CHCl2 (4.0 mL) and treated with
CF3CO2H (1.2 mL) to afford the free amine (22a; 141 mg, 88%) a
pale-yellow foam that collapsed into a translucent gum after
preparative TLC plate chromatography. TLC (CH2Cl2/MeOH 95:5):
Rf =0.50. NMR (400 MHz, DMSO): δ=8.53 (br s, 1H), 7.61 (d, J=
9.6 Hz, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.29 (dd, J=9.6, 2.0 Hz, 1H),
7.05 (d, J=8.8 Hz, 2H), 4.22 (s, 2H), 4.00 (t, J=5.6 Hz, 2H), 3.46 (q,
J=7.2 Hz, 2H), 3.38–3.24 (m, 4H), 2.94 (t, J=5.6 Hz, 2H), 1.18 (t, J=
7.2 Hz, 3H), 1.08 (t, J=7.2 Hz, 3H). 13C NMR (400 MHz, DMSO): δ=
167.0, 158.3, 143.4, 142.2, 129.0, 126.6, 124.7, 123.0, 118.4, 117.1,
116.4, 114.6, 69.6, 41.7, 40.6, 40.2, 28.7, 14.2, 13.1. IR (neat)max 3303
(w), 2973 (w), 2932 (w), 1699 (m), 1634 (m), 1521 (m), 1364 (m),
1330 (m), 1244 (s), 1170 (s), 1141 (m), 1097 (w), 1025 (m), 794 (w),
759 (w), 525 (w) cmÀ 1. MS (ESI+ve) m/z 401 (35Cl [M+H]+, 100%),
403 (37Cl [M+H]+, 33%). HRMS (ESI+ve TOF) calcd for
C21H2635ClN4O2 401.1736, found 401.1744 ([M+H]+).
Tert-butyl (2-(4-(6-chloro-3-(2-(diethylamino)-2-oxoethyl)
imidazo[1,2-a]pyridin-2-yl)phenoxy)ethyl)carbamate (21a)
Following General Procedure D, phenol (19; 250 mg, 0.70 mmol)
was dissolved in anhydrous DMF (10.5 mL) followed by sequential
addition of K2CO3 (387 mg, 28 mmol) and tert-butyl (2-bromoeth-
yl)carbamate (20a; 188 mg, 0.84 mmol) to afford the O-alkylated
phenol (21a; 250 mg, 71%) as a pale-yellow foam after prepara-
tive TLC plate chromatography. TLC (CH2Cl2/MeOH 95:5): Rf =0.74.
NMR (400 MHz, DMSO): δ=8.52 (br s, 1H), 7.60 (d, J=9.6 Hz, 1H),
7.52 (d, J=8.8 Hz, 2H), 7.28 (dd, J=9.6, 2.0 Hz, 1H), 7.05–7.01 (m,
3H), 4.20 (s, 2H), 4.01 (t, J=6.4 Hz, 2H), 3.45 (q, J=7.2 Hz, 2H),
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114.6, 99.2, 65.1, 41.7, 40.4, 40.1, 28.7, 27.5, 14.2, 13.1. IR (neat)max
3231 (w), 2967 (w), 1632 (m), 1582 (s), 1493 (s), 1464 (m), 1344 (s),
1284 (s), 1240 (m), 1136 (s), 1027 (m), 1009 (m), 802 (m), 740 (w),
596 (m), 525 (m) cmÀ 1. MS (ESI+ve) m/z 578 (35Cl [M+H]+, 100%),
580 (37Cl [M+H]+, 33%); (ESIÀ ve) m/z 576 (35Cl [MÀ H]À , 100%),
578 (37Cl [MÀ H]À , 33%). HRMS (ESI+ve TOF) calcd for
C28H2935ClN7O5 578.1913, found 578.1919 ([M+H]+).
2-(2-(4-(3-Aminopropoxy)phenyl)-6-chloroimidazo[1,2-a]
pyridin-3-yl)-N,N-diethylacetamide (22b)
1
2
3
4
5
Following General Procedure E, O-alkylated phenol (21b; 206 mg,
0.40 mmol) was dissolved in CHCl2 (4.0 mL) and treated with
CF3CO2H (1.2 mL) to afford the free amine (22b; 152 mg, 92%) a
pale-yellow foam that collapsed into a translucent gum after
preparative TLC plate chromatography. TLC (CH2Cl2/MeOH 95:5):
6
7
1
Rf =0.55. H NMR (400 MHz, DMSO): δ=8.51 (br s, 1H), 7.60 (d, J=
9.6 Hz, 1H), 7.53 (d, J=8.8 Hz, 2H), 7.27 (dd, J=9.6, 2.0 Hz, 1H),
7.02 (d, J=8.8 Hz, 2H), 4.21 (s, 2H), 4.06 (t, J=5.6 Hz, 2H), 3.46 (q,
J=7.2 Hz, 2H), 3.45–3.34 (m, 4H), 2.75 (t, J=5.6 Hz, 2H), 1.83
(quint, J=5.6 Hz, 2H), 1.17 (t, J=7.2 Hz, 3H), 1.05 (t, J=7.2 Hz, 3H).
13C NMR (400 MHz, DMSO): δ=167.0, 158.4, 143.5, 142.2, 129.0,
126.5, 124.7, 123.0, 118.4, 117.1, 116.4, 114.6, 65.5, 63.5, 41.7, 38.0,
31.6, 28.7, 14.2, 13.0. IR (neat)max 3367 (w), 2968 (w), 2932 (w), 1715
(w), 1632 (s), 1500 (m), 1389 (m), 1331 (m), 1246 (s), 1175 (m),
1141 (m), 1097 (w), 1025 (s), 795 (w), 761 (w), 526 (w) cmÀ 1. MS
(ESI+ve) m/z 415 (35Cl [M+H]+, 100%), 417 (37Cl [M+H]+, 33%).
HRMS (ESI+ve TOF) calcd for C22H2835ClN4O2 415.1895, found
415.1901 ([M+H]+).
8
9
2-(6-Chloro-2-(4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)oxy)
phenyl)imidazo[1,2-a]pyridin-3-yl)-N,N-diethylacetamide (24)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
The phenol (19; 175 mg, 0.49 mmol) was dissolved in DMF (4 mL)
°
with magnetic stirring. The solution was brought to 0 C (ice bath)
followed by sequential dropwise addition of DIPEA (256 μL,
1.47 mmol) and a solution of NBDÀ Cl (14; 98 mg, 0.49 mmol) in
DMF (1.0 mL) under a flow of N2 gas. The reaction vessel was then
sealed and allowed to warm to RT and stirring was continued for
18 h under N2 in the dark. The solution was partitioned between
CHCl3 (100 mL) and H2O (100 mL). The aqueous phase was
separated and extracted with CHCl3 (3×50 mL). The combined
organic extracts were washed with H2O (2×100 mL), brine (2×
100 mL), dried (Na2SO4), filtered, and concentrated. The residue
was subjected to flash chromatography over SiO2 gel (CH2Cl2/
MeOH 95:5) to afford the fluorescent probe (24; 110 mg, 43%) as
2-(6-Chloro-2-(4-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)
amino)ethoxy)phenyl)imidazo[1,2-a]
pyridin-3-yl)-N,N-diethylacetamide (23a)
°
a brown solid. TLC (CH2Cl2/MeOH 90:10): Rf =0.65. mp=98–100 C
(decomp). 1H NMR (400 MHz, DMSO): δ=8.67 (d, J=8.4 Hz, 1H),
8.58 (dd, J=2.0, 0.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 2H), 7.67 (dd, J=
9.6, 0.8 Hz, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.35 (dd, J=9.6, 2.0 Hz, 1H),
6.84 (d, J=8.4 Hz, 1H), 4.31 (s, 2H), 3.48 (q, J=7.2 Hz, 2H), 3.33 (q,
J=7.2 Hz, 2H), 1.20 (t, J=7.2 Hz, 3H), 1.07 (t, J=7.2 Hz, 3H). 13C
NMR (400 MHz, DMSO): δ=166.9, 153.1, 152.5, 145.4, 144.4, 142.4,
142.3, 135.5, 132.9, 130.4, 129.9, 125.3, 123.2, 121.0, 118.8, 117.6,
117.5, 110.0, 41.7, 40.2, 28.7, 14.2, 13.1. IR (neat)max 3404 (w), 2990
(w), 1632 (s), 1537 (s), 1521 (s), 1500 (s), 1450 (s), 1267 (s), 1257 (s),
1203 (s), 1088 (s), 1025 (s), 996 (s), 803 (s) cmÀ 1. MS (ESI+ve) m/z
521 (35Cl [M+H]+, 100%), 523 (37Cl [M+H]+, 100%); HRMS (ESI+
ve TOF) calcd for C25H2135ClN6O5 521.1335, found 521.1340 ([M+
H]+).
Following General Procedure C, amine (22a; 66 mg, 0.16 mmol)
was dissolved in DMF (3.2 mL) followed by sequential addition of
DIPEA (30 mg, 0.22 mmol) and NBDÀ Cl (14; 33 mg, 0.16 mmol) to
afford the desired fluorescent probe (23a; 40 mg, 44%) as a light-
brown solid after preparative TLC plate chromatography (CH2Cl2/
°
MeOH 98:2). TLC (CH2Cl2/MeOH 98:2): Rf =0.30. mp=102–104 C
(decomp). NMR (500 MHz, DMSO): δ=9.63 (br s, 1H), 8.56 (d, J=
9.0 Hz, 1H), 8.52 (s, 1H), 7.61 (d, J=9.5 Hz, 1H), 7.54 (d, J=9.0 Hz,
2H), 7.29 (dd, J=9.5, 2.0 Hz, 1H), 7.06 (d, J=9.0 Hz, 2H), 6.58 (d,
J=9.0 Hz, 1H), 4.36 (t, J=6.0 Hz, 2H), 4.20 (s, 2H), 3.92 (s, 2H), 3.45
(q, J=7.0 Hz, 2H), 3.31 (q, J=7.0 Hz, 2H), 1.17 (t, J=7.0 Hz, 3H),
1.07 (t, J=7.0 Hz, 3H). 13C NMR (500 MHz, DMSO): δ=167.0, 157.9,
145.3, 144.5, 144.1, 143.3, 142.2, 137.9, 129.0, 127.0, 124.8, 123.0,
121.2, 118.5, 117.2, 116.5, 114.7, 99.7, 65.6, 43.0, 41.7, 40.1, 28.7,
14.2, 13.1. IR (neat)max 3243 (w), 2976 (w), 1636 (m), 1583 (s), 1499
(s), 1444 (s), 1326 (m), 1292 (s), 1250 (s), 1128 (m), 1026 (m), 994
(m), 799 (m), 739 (m), 595 (m), 523 (m) cmÀ 1. MS (ESI+ve) m/z 564
(35Cl [M+H]+, 100%), 566 (37Cl [M+H]+, 33%); (ESIÀ ve) m/z 562
(35Cl [MÀ H]À , 100%), 564 (37Cl [MÀ H]À , 33%). HRMS (ESI+ve TOF)
calcd for C27H2735ClN7O5 564.1757, found 564.1762 ([M+H]+).
Biological studies
Animals. Male Sprague-Dawley rats and C57BL/6J mice were
purchased from Japan SLC (Shizuoka, Japan), kept in temparature-
controlled enviroment with a 12 h light-dark cycle, and fed a
standard diet (MB-1/Funabashi Farm, Chiba, Japan). Animals were
treated and handled according to the recommendations specified
by the Committee for the Care and Use of Laboratory Animals of
the National Institutes for Quantum and Radiological Science and
Technology (QST) and Animal Research: Reporting of In Vivo
Experiments (ARRIVE) guidelines. All animal experiments were
approved (approved number: 16-1006) by the committee of QST.
For in vitro fluorescence staining, six-months-old mice anesthe-
tized with 1.5% (v/v) isoflurane were injected with 2 μL of LSP in
saline (2 mg/mL) into striatum (Interaural 3.82 mm, Lateral
2.0 mm, Depth 2.5 mm) of right hemisphere via glass capillary.
2-(6-Chloro-2-(4-(3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)
amino)propoxy)phenyl)imidazo[1,2-a]
pyridin-3-yl)-N,N-diethylacetamide (23b)
Following General Procedure C, amine (22b; 80 mg, 0.19 mmol)
was dissolved in DMF (3.8 mL) followed by sequential addition of
DIPEA (34 mg, 0.27 mmol) and NBDÀ Cl (14; 38 mg, 0.19 mmol) to
afford the desired fluorescent probe (23b; 46 mg, 42%) as a light-
brown solid after preparative TLC plate chromatography (CH2Cl2/
In vitro binding assay. Four rats (8–10 weeks old) were sacrificed
by cervical dislocation under anesthesia (5% isoflurane in air). The
brains were rapidly removed and homogenized in ten volumes of
50 mM Tris·HCl (pH 7.4) containing 320 mM sucrose (Tris buffer)
using a Silent Crusher S homogenizer (Heidolph Instruments,
Schwabach, Germany). The homogenate was centrifuged in a
°
MeOH 98:2). TLC (CH2Cl2/MeOH 98:2). Rf =0.41. mp=105–107 C
(decomp). NMR (500 MHz, DMSO): δ=9.58 (br s, 1H), 8.52 (s, 1H),
8.49 (d, J=9.0 Hz, 1H), 7.60 (d, J=9.5 Hz, 1H), 7.53 (d, J=9.0 Hz,
2H), 7.28 (dd, J=9.5, 2.0 Hz, 1H), 7.04 (d, J=9.0 Hz, 2H), 6.45 (d,
J=9.0 Hz, 1H), 4.20 (s, 2H), 4.15 (t, J=6.0 Hz, 2H), 3.67 (br s, 2H),
3.44 (q, J=7.0 Hz, 2H), 3.32 (q, J=7.0 Hz, 2H), 2.17 (quint, J=
6.0 Hz, 2H), 1.16 (t, J=7.0 Hz, 3H), 1.06 (t, J=7.0 Hz, 3H). 13C NMR
(500 MHz, DMSO): δ=167.0, 158.2, 145.2, 144.5, 144.2, 143.4,
142.2, 137.9, 129.0, 126.7, 124.7, 123.0, 120.7, 118.4, 117.1, 116.4,
°
polypropylene tube at 800g for 10 min at 4 C. The supernatant
°
was collected and then centrifuged at 9000g for 10 min at 4 C
using an Optima-TLX (Beckman Coulter, Brea, CA). After discarding
the supernatant, the pellet was suspended in Tris-buffer and
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°
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1
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°
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sacrificed by cervical dislocation, and brains were subsequently
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°
fixed in 4% PFA in PBS for overnight at 4 C and autoclaved for
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Keywords: biological evaluations
·
fluorescent probes
imaging · neurodegenerative diseases · translocator protein
·
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Manuscript received: December 23, 2020
Revised manuscript received: February 22, 2021
Accepted manuscript online: February 25, 2021
Version of record online: ■■■, ■■■■
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Towards monitoring neuroinflam-
mation and -degeneration: A series
of TSPO-targeted probes based on 6-
chloro-2-phenylimidazo[1,2-a]
pyridine-3-yl acetamide ligands with
a NBD fluorophore has been
designed and synthesised. Their
efficacy was assessed in a competi-
tive binding assay on TSPO receptors
against [11C]PK11195. The probe with
the most potential can be considered
to be eligible for investigating
Dr. H. Wongso*, Dr. T. Yamasaki, Dr. K.
Kumata, Prof. M. Ono, Prof. M.
Higuchi, Prof. M.-R. Zhang, Prof. M. J.
Fulham, Prof. A. Katsifis*, Prof. P. A.
Keller*
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Design, Synthesis, and Biological
Evaluation of Novel Fluorescent
Probes Targeting the 18-kDa Trans-
locator Protein
activated microglia.